Milk provides essential nutrients to the newborn mammal. It also contains bioactive components for the management of gastrointestinal and other bodily functions, and for defense against microorganisms that can impact on health.
Lf is one of several bioactive components present in milk and colostrum. It is also present in most exocrine fluids, including tears and saliva. Lf has multiple biological roles including regulation of iron metabolism, immune function and embryonic development. Lf has anti-microbial activity against a range of pathogens including Gram-positive and Gram-negative bacteria, yeasts and fungi. The anti-microbial activity of Lf is due in part to its ability to bind the iron essential for the growth of certain bacteria. Lf also exerts bactericidal activity by binding to lipopolysaccharide (LPS) on bacterial membranes so disrupting the cell wall (Ellison et al, 1988). LPS are hydrophobic, negatively charged molecules also known as endotoxins. Lf may scavenge LPS from its environment during its isolation. Methods have been found to remove LPS from protein preparations (Franken at al, 2000; Petsch and Anspach, 2000; Ropp and Murray, 2006; Magalhaes et al, 2007) and from Lf (Rowe et al, 2006; Naidu, 2006 & 2008; Ward at al, 2009).
Lf has been proposed for use as an antimicrobial agent in the dairy and meat industries (Payne et al, 1990; Naidu, 2001). Natural Lf is partially saturated with iron (Reiter, 1985). Some researchers (Bishop et al, 1976; Korhonen, 1977; Payne et al, 1990.) reported that the antimicrobial activity of Lf depends on its iron saturation. Batish et al, (1988) found that the antibacterial activity of apo-Lf is greater than that of natural Lf and others have confirmed this.
Lf is an iron-binding glycoprotein with one iron-binding site in an N-terminus lobe and another in a C-terminus lobe. One molecule of Lf has the ability to bind reversibly to two high-spin Fe3+ ions in coordination with carbonate ions.
Domain opening is almost certainly the essential feature of iron release from Lf. There are three factors that trigger this process: i) interaction with specific Lf receptors, ii) reduction of the bound Fe3+ to Fe2+, and iii) reduced pH. Iron can be released from Lf by using water-soluble iron chelators and low pH (Groves, 1960; Masson and Heremans, 1968; Law and Reiter, 1977; Mazurier and Spik, 1980; Chung and Raymond, 1993; Feng, van der Does and Bantjes, 1993). However, completely removing iron is difficult and the iron saturation of apo-Lf is usually >10% (Batish et al, 1988; Payne et al, 1990; Chung and Raymond, 1993). Kontoghiorghes (1986) could not completely mobilize iron from Lf with any of a wide variety of soluble iron chelators at physiological pH due to the high affinity of Lf for iron (Aisen and Leibman, 1972; Chung and Raymond, 1993; Kretchmar Nguyen, Craig and Raymond, 1993). Some researchers used insoluble resins to chelate iron at pH<4 to prepare apo-Lf, but the apo-Lf still had an iron saturation of about 15% (Payne et al, 1990; Chung and Raymond, 1993). Although Feng, van der Does and Bantjes (1995) successfully removed iron from Lf with iron-chelating resin at physiological pH in the presence of citrate and other buffers, the method is complex, slow and the low Lf concentration used, which makes the process impractical for commercial use. Peterson at al (2000) showed that iron release does not begin until pH 3.5. Furthermore, because the iron removal processes were usually performed at pH<3.5 this lead to the development of turbidity in the solutions because of protein precipitation (Chung and Raymond, 1993). Modification of the conformation of the protein was sometimes observed (Mazurier and Spik, 1980).